Tenascin-C and alpha-smooth muscle actin positive cells are increased in the large airways in patients with COPD

Abstract

Background: Chronic obstructive pulmonary disease (COPD) is characterized by inflammation and remodeling of the lungs. This results in alterations in extracellular matrix (ECM) and structural changes leading to airflow obstruction. We studied the expression of tenascin-C (Tn-C) and alpha smooth muscle actin (α-SMA), which act as a marker of myofibroblasts, in large airways from COPD patients. Our aim was to elucidate whether this expression correlated with smoking or with disease development.

Methods: Bronchoscopy was performed on 20 COPD patients (mean age 56 years; range 39-61; FEV1/FVC < 70% and FEV1 median 53% (range 33-69) of predicted). Age and smoking matched smokers (S) without COPD (n = 13) and age matched non-smokers (NS) (n = 14) served as controls. Bronchial mucosal biopsies were analyzed by immunohistochemistry. The distribution of Tn-C expression was assessed and graded in three levels, and the number of spindle shaped cells staining positive for α-SMA were counted.

Results: Biopsies from COPD patients had more (P < 0.001) Tn-C expression than the two control groups. A significantly (P < 0.05) increased number of spindle shaped cells expressing α-SMA was observed in COPD patients compared with the controls. Smokers and nonsmokers did not differ in this respect. The expression of Tn-C correlated positively (P < 0.001) to the number of α-SMA positive cells.

Conclusions: We demonstrate increased expression of Tn-C and α-SMA positive cells in the large airways in COPD. This was not associated to smoking per se, but to the presence of airway obstruction. Our findings add new information regarding remodeling characteristics and highlight the large airways as a potential site for airways obstruction in COPD.

Figure 1

The immunohistochemical expression of Tn-C (1 A-C) and α-SMA (1 D-F) in bronchial biopsies from study subjects. The figures show representative microphotographs from each category. A: Positivity for Tn-C is seen in tangentially sectioned basal epithelial cells and along the basement membrane (BM) of bronchial epithelium (arrows); scale bar = 0.05 mm. B: Tn-C positivity in basal epithelial cells and in the stroma underneath the BM (arrows); scale bar = 0.05 mm. C: Positivity for Tn-C in basal epithelial cells and in a wide area of stromal connective tissue underneath the BM (arrows); scale bar = 0.05 mm. D: Spindle shaped cells positive for α-SMA (arrows) in a biopsy graded to the category 1-4 positive cells. Smooth muscle of the bronchial wall (SM) or blood vessels (arrow heads) were not counted; scale bar = 0.05 mm. E: A bronchial biopsy graded to the category 5-10 α-SMA positive cells (arrows). Blood vessels (arrow heads) or smooth muscle layer of the bronchial wall (SM) were not counted; scale bar = 0.05 mm. F: More than 10 spindle shaped cells are showing positivity for α-SMA (arrows) in a bronchial biopsy; scale bar = 0.05 mm.

Figure 2

Number and proportion of subjects expressing Tenascin C outside the basal epithelial cells and basement membrane. Data is given for the three groups COPD, smoking controls (S) and non-smoking controls (NS). Tn C (b+c): All subjects with expression outside the basal epithelial cells and basement membrane. Tn C^©: Subjects with expression within connective tissue beyond the stroma underneath basal membrane. The number (n) of individuals in each category is presented underneath corresponding bar. The Odds ratio for a COPD patient to be in a higher category is statistically increased (P < 0.001) compared to subjects in the control groups.

Figure 3

The immunohistochemical expression of α-SMA, desmin and vimentin in bronchial biopsies from study subjects. The figures show representative microphotographs from each category. A: High power field of spindle shaped cells positive for α-SMA; scale bar = 0.05 mm. B: High power field of desmin positive spindle shaped cells; scale bar = 0.05 mm. C: Ring like structures of blood vessels positive for α-SMA; scale bar = 0.05 mm. D: Thick bundles of smooth muscle of the bronchial wall, staining for desmin; scale bar = 0.05 mm. E: Staining for vimentin from a case in which no α-SMA or desmin positive spindle shaped cells were found. Positive staining pattern in normal fibroblasts and lymphocytes of the subepithelial connective tissue; scale bar = 0.05 mm. F: A Negative control in which the primary antibody has been substituted with non-immune mouse serum; scale bar = 0.1 mm.

Figure 4

Number and proportions of subjects with cells staining positive for α-SMA. Data is given for the three groups COPD, smoking controls (S) and non-smoking controls (NS). The numbers (n) of individuals in each category is presented as digits underneath each bar. The odds for a COPD patient to be in a higher category is statistically increased (P < 0.05) compared to subjects in the control groups.

Figure 5

Correlation between staining for Tenascin C and α-SMA in all subjects. Increasing degree of staining is indicated by categories a-c and a-d. Number in the circles indicate number of subjects. The estimate for the correlation coefficient was 0.6; P < 0.0001.